Linkingenergyproduction and proteinsynthesis in hydrogenotrophicmethanogens.

1Interdepartmental Program in Biomolecular Science and Engineering, University of California, Santa Barbara, Santa Barbara, California 93106-9510, United States.

Abstract

Hydrogenotrophicmethanogens possessing the hydrogen-dependent dehydrogenase Hmd also encode paralogs of this protein whose function is poorly understood. Here we present biochemical evidence that the two inactive Hmd paralogs of Methanocaldococcus jannaschii, HmdII and HmdIII, form binary and ternary complexes with several components of the protein translation apparatus. HmdII and HmdIII, but not the active dehydrogenase Hmd, bind with micromolar binding affinities to a number of tRNAs and form ternary complexes with tRNA(Pro) and prolyl-tRNA synthetase (ProRS). Fluorescence spectroscopy experiments also suggest that binding of HmdII and ProRS involves distinct binding determinants on the tRNA. These biochemical data suggest the possibility of a regulatory link between energyproduction and protein translation pathways that may allow a rapid cellular response to altered environmental conditions.

Size exclusion HPLC coupled to multiangle light scattering. Superimposed runs for ProRS, Hmd and Hmd paralogs are shown color-coded as indicated. The plot depicts the logarithm of the molecular weight on the ordinate and the elution time on the abscissa. The second ordinate depicts the refractive index gradient. Molecular weights were determined from the light scattering data.

Formation of Hmd-tRNAPro binary complexes measured by steady-state fluorescence anisotropy for Hmd (panel B), HmdII (panel C) and HmdIII (panel D). The fluorescent label is present as 5’-fluorescein conjugated tRNAPro. The control experiment using the identical methodology to measure the affinity of the ProRS-tRNAPro complex is shown in panel A.

EMSA depicting binary complex formation between Hmd proteins and tRNAPro. Native PAGE show the mobility shift of 3'-labeled tRNA upon binding as Hmd (A), HmdII (B), or HmdIII (C) are titrated in. The fraction of bound tRNA is shown as a function of the dimeric concentration of each protein.

(A) Titration of Hmd into the preformed ProRS-tRNAPro complex, observed by fluorescence anisotropy. The fluorescent label is present as 5’-fluorescein conjugated tRNAPro. (B) EMSA for complex formation among ProRS, tRNAPro and Hmd. While ProRS-tRNAPro complex formation is observed, only a weak mobility shift of radiolabeled tRNA occurs for the Hmd binary complex. No change in the tRNA gel mobility shift generated by ProRS is observed upon further titration of Hmd. (C) Ternary complex formation for ProRS:tRNAPro:HmdII observed by fluorescence polarization. The binding events are observed as ProRS (red circles) is first titrated into 5’-fluorescein conjugated tRNAPro; a second binding event is observed upon further titration of HmdII (blue circles). (D) Ternary complex formation observed by fluorescence polarization for ProRS:tRNAPro:HmdIII depicted identically to panel (C). (E) Formation of the ProRS-tRNAPro-HmdII ternary complex observed by EMSA. tRNAPro is radiolabeled with 32P as described in Methods. Supershifting upon addition of HmdII is evident. Similar data for HmdIII is not shown. (F) SDS PAGE gel of samples excised from the nondenaturing gel depicted in panel (E). Lanes A, B, C on this gel correspond to samples excised from the corresponding positions in the native gel shown in panel (E).

Complex formation observed by steady-state fluorescence quenching. 5’- fluorescein conjugated tRNAPro is used in these experiments. Titration of ProRS (red and orange traces) produces a significant quenching signal, while titration of HmdII does not.

(A) Sequence alignment of Mj Hmd and Mj HmdII. Amino acids that are conserved among the HmdII and HmdIII paralogs from all hydrogenotrophicmethanogens are shaded in orange, while those conserved among all active Hmd enzymes are shaded in green. Basic residues found in the C-terminal extension that is unique to the paralogs, and that are conserved in over 75% of paralog sequences, are highlighted in blue. In this alignment of Mj Hmd sequences, a sixth basic residue (not highlighted) is present that is conserved in 50% of the other HmdII and HmdIII sequences. (B) Crystal structure of Hmd (RCSB code 3DAG).21 Amino acids highlighted in panel (A) that are unique to the paralogs or to active Hmd enzymes are displayed in orange and green, respectively, on the tertiary structure. A depiction of the C-terminal peptide for illustrative purposes is shown in light blue. The position of this peptide with respect to the remainder of the enzyme structure has not been modeled. The peptide is shown containing two α-helices for consistency with earlier structure predictions.20,24